In the proposed study we plan to investigate further the structural and mechanistic features of calcium transport carried out by the ATPase of sarcoplasmic reticulum membranes. The two principal approaches will utilize (1) non-covalent (reversibly bound) probes, such as ATP analogs, and (2) covalent (irreversibly bound) modification, primarily directed towards specific labeling of the nucleotide sites. Determination of the nature and extent of interactions will be done primarily by spectrophotometric, spectrofluorimetric, and enzyme activity measurements. Among the specific parameters to be obtained are stoichiometric, binding strength, and cooperativity constants associated with nucleotide and calcium binding and how these change during the calcium transport cycle; and distances between sites (measured by fluorescence energy transfer). From covalent modification experiments, we expect to identify further the functional groups which participate in substrate binding and catalysis, and to detect protein conformation changes which occur during the calcium transport cycle.